Alpha-amylases are widely distributed throughout nature. The activity of .alpha.-amylase is characterized by the hydrolysis of .alpha.-1,4 glucosidic bonds in an endo-fashion, in a process known industrially as liquefaction. Due to the high viscosities and mass transfer problems, industrial liquefaction is carried out at the highest possible temperature. Typically, using the .alpha.-amylase from B. licheniformis, the starch slurry along with calcium ions are incubated at 100.degree. C. for 5 to 10 minutes then kept at 95.degree. C. for 1 to 2 hours (Ng, T. K., and W. F. Keneally in Thermophiles: General, Molecular and Applied Microbiology, T. D. Brock, ed., John Wiley & Sons, New York (1986)). This process is essential for several industries. Glucopolymers with varying degrees of polymerization are utilized in papermaking, textile preparation, brewing and fermentation. Liquefaction is a preliminary step in saccharification by which large quantities of D-glucose can be produced from inexpensive sources.
Thermostable .alpha.-amylases are well suited for the elevated temperatures used in industrial liquefaction in that the more thermostable the enzyme, the more efficient the hydrolysis at extreme temperature. The enzymes isolated from thermophilic microorganisms display inherent thermostability comparable to their optimal growth temperature. Thus, the higher the optimal growth temperature of the microorganism, the greater the likelihood that it will produce an .alpha.-amylase suitable for industrial application.
Many .alpha.-amylases have been purified which display thermostable characteristics. These range from moderate thermophiles, with optimal enzymatic activities within the range of 50.degree. to 80.degree. C. (Antrankian, Applied Biochem. Biotech. 20/21: 267 (1989), Glymph and Stutzenberger, Applied Environ. Microbiol., 34: 391 (1977), Hasegawa et al, J. Biochem, 79: 35 (1976)), to hyperthermophiles with optimal activities above 80.degree. C. (Schumann et al, FEBS, 282: 122 (1991), Koch et al, Arch. Microbiol, 155: 572 (1991), Laderman et al (1992)). .alpha.-Amylases isolated from microorganisms with optimal growth temperatures at or near 100.degree. C. have been found to have optimal activities at this temperature, with residual activity at temperatures as high as 120.degree. C., making them ideally suited for industrial application.
Using genetic engineering technology, it is theoretically possible to clone genes and produce the enzymes that they encode in quantities sufficient for industrial application. A number of genes coding for thermophilic .alpha.-amylases have been isolated and subsequently expressed in E. coli and B. subtilis (Fukusumi et al, Eur. J. Biochem., 98: 95 (1985), Tsukagoshi et al, Mol. Gen. Genet., 195: 58 (1984), Tsukagoshi et al, J. Bacteriology, 164: 1182 (1985)). The temperature at which the genes are endogenously translated does not seem to have an effect on the expression in transformation competent cells. Thus it is possible to produce thermophilic enzymes in host cells grown at ambient temperature. However, no genes coding for hyperthermophilic .alpha.-amylases have ever been successfully cloned.
The hyperthermophilic archaebacterium Pyrococcus furiosus has been isolated from solfateric mud and found to have an optimal growth temperature of 100.degree. C. (Fiala and Stetter, Arch. Microbiol., 145: 56 (1968)). .alpha.-Amylase activity from P. furiosus has been detected in the cell-free supernatant (Antrankikian et al, WO 90/11352, (1990)), and in crude cell extract (Brown et al, Appl. Environ. Microbiol., 56: 1985 (1990)). The .alpha.-amylase derived from this bacterium is known to be stable at high temperature and is expected to be widely used in industrial fields. However, methods for the industrial production of this enzyme from P. furiosus are not established and neither the gene structure nor the amino acid sequence of this enzyme has been reported.
The present invention provides, for the first, time a cloned sequence encoding a hyperthermophilic .alpha.-amylase. The availability of this sequence makes possible the industrial scale production of this enzyme.